Project Summary Helicobacter pylori (H. pylori) is a gram-negative bacterial pathogen that colonizes the human stomach. H. pylori colonization in humans has been associated with several stomach illnesses, ranging from chronic gastritis, ulceration, and adenocarcinoma. The cytotoxin-associated gene-pathogenicity island encodes a type IV protein secretion system (cag PAI T4SS) that is known to play an essential role in the development and progression of the diseases. This cag PAI T4SS functions to inject virulence bacterial effector protein CagA into the host cell. Extensive studies have been carried out to characterize how CagA functions to promote gastric adenocarcinomas and bacterial virulence. However, a wide range of host cellular responses are type IV-dependent, but independent of CagA, making it likely that additional bacterial effectors exist for the cag PAI T4SS. Type III effector proteins are readily detected in the culture supernatants even though they are known to exert their functions inside the infected host cells. The pertussis toxin is a T4SS substrate that is secreted into the extracellular milieu as opposed to being injected across host-cell membranes. Despite intensive efforts, no one has been able to detect the type IV effectors from the H. pylori culture supernatants and this has led to the assumption that type IV effectors are directly injected into host cells during H. pylori infection. However, we found CagA in H. pylori-infected supernatants in a type IV-dependent manner, indicating that type IV effectors may be leaked into the infected media by H. pylori. We have employed a series of advanced biochemical and cellular screening procedures to identify novel H. pylori T4SS effectors during Helicobacter infection of gastric epithelial cells. We have discovered additional type IV effectors in additional to CagA. Using three independent methods, we have validated that Hp367 is indeed translocated into the host cell via a cag PAI T4SS-dependent manner. Thus, in addition to CagA, Hp367 is a bona fide cag PAI T4SS-dependent effector during H. pylori infection. In this proposal, we plan to 1) Identify and validate type IV effectors; 2) Assess the phenotypes of the effector mutants and their roles in inflammation. Results from our study will fundamentally change the current view that CagA is the only type IV effect in H. pylori. New effectors identified from our study may open up new research fronts in Helicobacter to better understand how H. pylori induces ulcer and even cancer during infection.